Project Summary/Abstract We aim to develop a new therapeutic strategy for one major form of JAK2V617F-induced myeloproliferative neoplasms (MPNs), polycythemia vera (PV), with a focus on targeting PV-initiating cells (cancer stem cells in PV). A curative therapy for PV is still lacking. The proposed studies are based on our preliminary finding that the arachidonate 5-lipoxygenase (5-LO) gene (Alox5) plays a key role in survival regulation of PV-initiating cells. The Alox5 pathway is known for producing inflammatory (asthma-causing) leukotrienes, and an FDA-approved human anti-asthma drug called Singulair (Montelukast) blocks leukotriene receptor binding to reduce the inflammatory response. We have found that Singulair inhibits the growth of JAK2-V617F-expressing cells in vitro and in mice. Thus, Singulair could be an effective new agent for treating PV. We hypothesize that leukotrienes produced in the Alox5-pathway play an essential role in survival regulation of MPN-initiating cells in PV, and Singulair antagonizes the action of leukotrienes to act as a potential new therapeutic agent for PV. Testing this hypothesis is of clinical significance and would benefit: 1) PV patients who have not progressed to acute myeloid leukemia (AML); 2) PV patients who are in clinical remission of AML with residual JAK2V617F-expressing cells and may relapse with time; 3) PV patients who had bone marrow transplantation for treating AML progressed from PV but still have residual JAK2V617F-expressing cells; and 4) the individuals who have detectable JAK2V617F transcripts in myeloid cells but have not developed clinical symptoms of PV. Because the Alox5 pathway is known to produce leukotrienes, one plausible mechanistic explanation of action of Singulair is that it eradicates PV by blocking receptor binding of leukotriens to PV-initiating cells. We also need to know the underlying molecular mechanisms by which Singulair inhibits PV-initiating cells and PV development. In fact, our preliminary studies show that JAK2V617F regulates the Alox5 pathway involving two Alox5 downstream genes, beta-catenin (as a stimulator) and Blk (as a suppressor), and it will be important to investigate whether Singuliar regulates these two genes in PV-initiating cells. Finally, it is critical to determine whether Singulair inhibits the growth of human PV-initiating cells. The specific aims are: 1) To determine whether the survival of PV-initiating cells is dependent on leukotrienes and whether a blockade of leukotrienes by Singulair reduces their effects on PV-initiating cells; 2) To determine the molecular mechanisms by which Singulair inhibits PV-initiating cells and PV development; and 3) To determine whether Singulair blocks the Alox5 pathway in human PV-initiating cells and reduces engraftment of human PV-initiating cells in immunocompromised mice. Broadly, Singulair is a potential therapeutic agent for solid tumors as Alox5 is associated with tumorigenesis in the colon, lung, pancreas, liver, esophagus, skin, etc.